Acyclovir biodegradation followed cometabolism by AOB in the presence of ammonium. Alcohol oxidation was the main biotransformation pathway producing carboxy-acyclovir. Formation of carboxy-acyclovir was independent on the initial concentration of acyclovir. Metabolic type has no effect on the biotransformation pathway into carboxy-acyclovir. The enzyme-linked oxidation could be catalyzed by AOB or heterotrophs.